Cancer treatment

ABSTRACT

The present invention relates to compositions, methods, uses and kits for treating cancer. The present invention relates to methods for minimising the progression of cancer in a subject, the method comprising administering to the subject therapeutically effective amounts of chymotrypsinogen and trypsinogen, thereby minimising the progression of cancer in the subject. In particular, the methods provide a means for treating cancer by reducing the number of cancer stem cells in the subject.

FIELD OF THE INVENTION

The present invention relates to compositions, methods, uses and kitsfor treating cancer.

Cross Reference to Applications

This application claims priority from Spanish patent applications, P201630112 and P 201631662, the entire contents of which are hereinincorporated in their entirety.

BACKGROUND OF THE INVENTION

Despite improvements in therapies for the treatment of cancer, thecancer mortality rate worldwide remains high and strategies to preventcancer recurrence are still needed. Current therapeutic strategiesagainst cancer frequently result in treatment failure, often due to thedevelopment of multiple malignancies and/or resistance to chemotherapyand radiotherapy.

Cancer stem cells (CSCs) are immortal tumour-initiating cells that havethe capacity to self-renew and have pluripotent capacity (Reya et al.,(2001). Nature 414:105-111). CSCs are found in multiple malignancies,including leukaemia and many solid tumours and, given their stem-celllike properties, are thought to be the basis for tumour initiation,development, metastasis and recurrence (Tang DG. Cell Res 2012; 22:457-72).

CSCs represent only a small fraction of the cancer cells within a tumourand can remain quiescent for extended periods of time, thereby evadingconventional therapies (e.g., chemotherapy and radiotherapy) that aretargeted to highly proliferative cells (Chikamatsu et al., (2011) HeadNeck. 34(3):336-43). Consequently, a priority for improving cancertreatment and reducing the risk of cancer relapse is to develop newstrategies that selectively target CSC eradication while sparing normalstem cells.

Reference to any prior art in the specification is not an acknowledgmentor suggestion that this prior art forms part of the common generalknowledge in any jurisdiction or that this prior art could reasonably beexpected to be understood, regarded as relevant, and/or combined withother pieces of prior art by a skilled person in the art.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method ofminimising the progression of cancer in a subject who has received atreatment for cancer, the method comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen,thereby minimising the progression of cancer in the subject.

In a further aspect, the invention relates to a method of treatingminimal residual disease in a subject who has received a treatment forcancer, the method comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen,thereby treating minimal residual disease in a subject.

In a further aspect, the invention relates to a method of preventingminimal residual disease in a subject who is receiving a treatment forcancer, the method comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen,thereby preventing minimal residual disease in a subject.

In a further aspect, the invention relates to a method of preventingrecurrence of cancer in a subject who has received a treatment forcancer, the method comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen,thereby preventing recurrence of cancer in the subject.

In a further aspect, the invention relates to a method of preventing orinhibiting metastasis of cancer in a subject, the method comprisingadministering to the subject therapeutically effective amounts ofchymotrypsinogen and trypsinogen, thereby preventing metastasis ofcancer in the subject.

In yet a further aspect, the invention relates to a method ofsensitising a subject for subsequent treatment for cancer, the methodcomprising administering a therapeutically effective amount ofchymotrypsinogen and trypsinogen to the subject before the subjectreceives treatment for cancer, thereby sensitising the subject forsubsequent treatment for cancer.

In yet a further aspect, the invention relates to a method of preventingcancer in a subject, the method comprising administering atherapeutically effective amount of chymotrypsinogen and trypsinogen tothe subject, wherein the subject is considered at risk of thedevelopment of cancer.

The invention also relates to a method of delaying the onset of cancerin a subject, the method comprising administering therapeuticallyeffective amounts of chymotrypsinogen and trypsinogen, thereby delayingthe onset of cancer in the subject.

In any method or use of the invention, the number of cancer stem cellsin the subject may be reduced or prevented from increasing in number.Alternatively, the cancer stem cells may be differentiated.Identification of cancer stem cells or the extent of differentiation maybe determined by any method described herein using markers as describedherein, particularly Table 1.

Further provided are compositions for minimising the progression ofcancer in a subject who has received a treatment for cancer, comprisingchymotrypsinogen and trypsinogen.

In another aspect, the invention provides compositions for treatingminimal residual disease in a subject who has received a treatment forcancer, comprising chymotrypsinogen and trypsinogen.

In still a further aspect, the invention provides compositions forpreventing metastasis of cancer in a subject, comprisingchymotrypsinogen and trypsinogen.

The invention also contemplates the use of chymotrypsinogen andtrypsinogen in the manufacture of a medicament for minimising theprogression of cancer in a subject, wherein the subject has previouslyreceived a treatment for cancer.

Still further, the invention contemplates the use of chymotrypsinogenand trypsinogen in the manufacture of a medicament for treating minimalresidual disease in a subject who has received a treatment for cancer.

In any method of the invention described herein, the method may furtherinclude the step of identifying the presence of cancer stem cells in thesubject.

In any method of the invention described herein, the method may furtherinclude the step of providing a subject who has received a treatment forcancer. Further, the subject may not have detectable cancer at the timethat the chymotrypsinogen and trypsinogen is administered, for example,the cancer may have substantially diminished in size, mass or otherphysical measure as a consequence of the prior treatment at the timethat the chymotrypsinogen and trypsinogen is administered to thesubject.

The invention also relates to a method of treating cancer in a subject,the method comprising the steps of:

-   -   identifying the presence of cancer stem cells in the subject;        and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen, thereby treating cancer in the        subject.

The invention also relates to a method of preventing cancer in asubject, the method comprising the steps of:

-   -   identifying the presence of cancer stem cells in the subject who        has not been diagnosed as having cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing cancer in the subject.

The invention also relates to a method of preventing cancer in asubject, the method comprising the steps of:

-   -   providing a subject identified as being at risk of developing        cancer but who has not been diagnosed as having cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen, thereby preventing cancer in        the subject.

The invention also relates to a method of delaying the onset of cancerin a subject, the method comprising the steps of:

-   -   providing a subject at risk of cancer but who has not been        diagnosed as having cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby delaying the onset of cancer in the subject.

In any embodiment of the present invention, a subject or individual whois considered at risk of cancer may be an individual who has a familyhistory of cancer, have one or more biomarkers associated with cancer,including having cancer stem cells.

As such, the present invention also relates to a method of preventingcancer in a subject, the method comprising the steps of:

-   -   identifying the presence of cancer stem cells in the subject,        wherein the subject has not been diagnosed as having cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing cancer in the subject.

The invention also relates to a method of preventing cancer in asubject, the method comprising the steps of:

-   -   identifying the presence of cancer stem cells in the subject who        is at risk of developing cancer but has not been diagnosed as        having cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing cancer in the subject.

The invention also relates to a method of delaying the onset of cancerin a subject, the method comprising the steps of:

-   -   identifying the presence of cancer stem cells in the subject who        is at risk of developing cancer but has not been diagnosed as        having cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby delaying the onset of cancer in the subject.

In a further embodiment, the invention relates to a method of preventingthe recurrence of cancer in a subject, the method comprising the stepsof:

-   -   providing an individual who has received a treatment for cancer;        and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing the recurrence of cancer in the subject.

In a further embodiment, the invention relates to a method of preventingmetastasis of cancer in a subject, the method comprising the steps of:

-   -   providing an individual who is to receive a treatment for        cancer, who is receiving a treatment of cancer, or who has        received a treatment for cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing metastasis of cancer in the subject.

Yet further, the invention relates to a method of delaying therecurrence of cancer in a subject, the method comprising the steps of:

-   -   providing an individual who has received a treatment for cancer;        and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby delaying the recurrence of cancer in the subject.

Still further, the invention relates to a method of delaying the onsetof cancer in a subject, the method comprising the steps of:

-   -   providing an individual who is at risk of the development of        cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby delaying the onset of cancer in the subject.

Preferably, identifying the presence of cancer stem cells occurs at thesite of an existing tumour or at the site of therapeutic intervention.Typically, the therapeutic intervention is surgical resection,chemotherapy or radiotherapy.

Preferably, cancer stem cells are identified using any one or moremarkers as described herein such as in Table 1. For example,pancreatic-specific CSC markers include CD326, CD44 and CxCR4 andcolon-specific CSC markers: CD326 and CD44 and analysed byimmunofluorescence or flow cytometry.

The invention also relates to a method of treating cancer in a subject,the method comprising the steps of:

-   -   identifying the presence of cancer stem cells in the subject by        determining the presence of any one or more of the cell surface        markers in Table 1; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby treating cancer in the subject.

The invention also relates to a method of treating colon cancer in asubject, the method comprising the steps of:

-   -   identifying the presence of colon cancer stem cells in the        subject by determining the presence of any one or more of the        cell surface markers CD326 and CD44; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby treating colon cancer in the subject.

The invention also relates to a method of preventing colon cancer in asubject, the method comprising the steps of:

-   -   identifying the presence of colon cancer stem cells in the        subject by determining the presence of any one or more of the        cell surface markers CD326 and CD44; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing colon cancer in the subject.

The invention also relates to a method of delaying the onset of coloncancer in a subject, the method comprising the steps of:

-   -   identifying the presence of colon cancer stem cells in the        subject by determining the presence of any one or more of the        cell surface markers CD326 and CD44; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby delaying the onset of colon cancer in the subject.

The invention also relates to a method of preventing the recurrence ofcolon cancer in a subject, the method comprising the steps of:

-   -   providing a subject who has received a treatment for colon        cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing the recurrence of colon cancer in the subject.

The invention also relates to a method of sensitising a subject to atreatment for colon cancer, the method comprising the steps of:

-   -   providing a subject who is to receive a treatment for colon        cancer and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby sensitising the subject for a treatment for colon cancer.

The invention also relates to a method of treating pancreatic cancer ina subject, the method comprising the steps of:

-   -   identifying the presence of pancreatic cancer stem cells in the        subject by determining the presence of any one or more of the        cell surface markers CD326, CD44 and CxCR4; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby treating pancreatic cancer in the subject.

The invention also relates to a method of preventing pancreatic cancerin a subject, the method comprising the steps of:

-   -   identifying the presence of pancreatic cancer stem cells in the        subject by determining the presence of any one or more of the        cell surface markers CD326, CD44 and CxCR4; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing pancreatic cancer in the subject.

The invention also relates to a method of delaying the onset ofpancreatic cancer in a subject, the method comprising the steps of:

-   -   identifying the presence of pancreatic cancer stem cells in the        subject by determining the presence of any one or more of the        cell surface markers CD326, CD44 and CxCR4; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby delaying the onset of pancreatic cancer in the subject.

The invention also relates to a method of preventing the recurrence ofpancreatic cancer in a subject, the method comprising the steps of:

-   -   providing a subject who has received a treatment for pancreatic        cancer; and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby preventing the recurrence of pancreatic cancer in the subject.

The invention also relates to a method of sensitising a subject to atreatment for pancreatic cancer, the method comprising the steps of:

-   -   providing a subject who is to receive a treatment for pancreatic        cancer and    -   administering therapeutically effective amounts of        chymotrypsinogen and trypsinogen,

thereby sensitising the subject for a treatment for pancreatic cancer.

The present invention also relates to a composition comprises orconsists of chymotrypsinogen and trypsinogen for use in (a) minimisingthe progression of cancer in a subject who has received a treatment forcancer, (b) treating minimal residual disease in a subject who hasreceived a treatment for cancer, or (c) of sensitising a subject forsubsequent treatment for cancer. Typically, the composition furthercomprises a pharmaceutically acceptable diluent, excipient or carrier.

The invention also provides a kit minimising the progression of cancerin a subject who has received a treatment for cancer, comprising atleast one dosage unit, wherein the dosage unit compriseschymotrypsinogen, trypsinogen and a pharmaceutically acceptable diluent,excipient or carrier. Optionally the kit also includes writteninstructions directing the user to administer a dosage unit ofchymotrypsinogen according to a method of the invention describedherein.

The invention also provides a kit of the invention when used in a methodof the invention.

In any aspect of the invention, chymotrypsinogen and trypsinogen isadministered in a weight ratio in the range of at or about 1: 1 to at orabout 10: 1, at or about 4: 1 to at or about 8: 1, at or about 5: 1 toat or about 7: 1, or at about 6: 1. Further, any composition describedabove has chymotrypsinogen and trypsinogen in a weight ratio in therange of at or about 1: 1 to at or about 10: 1, at or about 4: 1 to ator about 8: 1, at or about 5: 1 to at or about 7: 1, or at about 6: 1.

In any aspect of the invention, chymotrypsinogen and trypsinogen isadministered intravenously, subcutaneously or intramuscularly.

In any aspect of the invention, chymotrypsinogen and trypsinogen may beadministered simultaneously or sequentially.

In any aspect of a method or use of the invention, the method or usefurther comprises the step of identifying a subject having, or at riskof developing, cancer. Preferably, the cancer is any one describedherein.

In any aspect of the invention, the composition does not contain or themethod or use does not administer, amylase.

In any aspect, embodiment or form of the invention described herein theamount of chymotrypsinogen administered may be greater than, or equalto, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 3.5 mg/kg, 5 mg/kg, 15 mg/kg, 20 mg/kg,40 mg/kg, 45 mg/kg, 135 mg/kg, 250 mg/kg or 500 mg/kg.

In any aspect, embodiment or form of the invention described herein theamount of trypsinogen administered may be greater than, or equal to, 0.2mg/kg, 0.25 mg/kg, 0.4 mg/kg, 0.6 mg/kg, 0.8 mg/kg, 2 mg/kg, 2.5 mg/kg,3 mg/kg, 5 mg/kg, 7 mg/kg, 8 mg/kg, 20 mg/kg, 40 mg/kg or 80 mg/kg.

In any aspect, embodiment or form of the invention described herein theamount of chymotrypsinogen administered to a human may be greater than,or equal to, 0.1 mg/kg, 0.15 mg/kg, 0.25 mg/kg, 0.4 mg/kg, 1.2 mg/kg,3.5 mg/kg, 10 mg/kg, 20 mg/kg or 40 mg/kg.

In any aspect, embodiment or form of the invention described herein theamount of trypsinogen administered to a human may be greater than, orequal to, 0.02 mg/kg, 0.03 mg/kg, 0.05 mg/kg, 0.06 mg/kg, 0.2 mg/kg, 0.6mg/kg, 1.5 mg/kg, 3 mg/kg or 6 mg/kg.

Preferably, in any aspect, embodiment or form of the invention describedherein the amount of chymotrypsinogen administered may be in the rangeof 1 mg/kg to 41 mg/kg, 1.5 mg/kg to 500 mg/kg, 2 mg/kg to 250 mg/kg,3.5 mg/kg to 135 mg/kg, 5 mg/kg or 15 mg/kg to 45 mg/kg.

Preferably, in any aspect, embodiment or form of the invention describedherein the amount of trypsinogen administered may be greater than 0.2mg/kg to 7 mg/kg, 0.25 mg/kg to 80 mg/kg, 0.4 mg/kg to 40 mg/kg, 0.6mg/kg to 20 mg/kg, 0.8 mg/kg to 8 mg/kg, or 2.5 mg/kg to 8 mg/kg.

In any composition of the invention above, the composition may beadapted to administer the relevant mg, or mg/kg, of chymotrypsinogen andtrypsinogen to the subject.

As used herein, except where the context requires otherwise, the term“comprise” and variations of the term, such as “comprising”, “comprises”and “comprised”, are not intended to exclude further additives,components, integers or steps.

Further aspects of the present invention and further embodiments of theaspects described in the preceding paragraphs will become apparent fromthe following description, given by way of example and with reference tothe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : MTT assay to determine IC₅₀ for Trypsinogen/Chymotrypsinogen.Rate of cell proliferation vs treatment concentration is shown for colonHCT-116 CSCs and pancreatic BxPC3 CSCs treated with varyingconcentrations of Trypsinogen/Chymotrypsinogen (1:6) or interferon alpha(IFN).

FIG. 2 : Cell cycle assay for human pancreatic cancer cell line BxPC3.Panels a and b show assay results for pancreatic non-CSCs; panels c andd show assay results for pancreatic CSCs.

FIG. 3 : Cell cycle assay for human colon cancer cell line HCT-116.Panels a and b show assay results for colon non-CSCs; panels c and dshow assay results for colon CSCs.

FIG. 4 : Aldehyde dehydrogenase assay for human pancreatic cell lineBxPC3. The percentage of ALDH-positive cells in pancreatic non-CSCs andCSCs is shown following treatment of the cells with control, PRP,PRP+IFN or IFN.

FIG. 5 : Dot plot results of flow cytometry sorting of pancreatic CSCson the basis of cell-surface expression of markers CD 326, CD 44 andCxCR4 following treatment of the cells with PRP, PRP+IFN or IFN.

FIG. 6 : Histogram showing expression of CSC markers in pancreatic CSCs:percentage of cells having the three markers CD326, CD44, CxCR4 areshown for CSCs treated with PRP, PRP+IFN or IFN and for non-CSCs. Theresults of two separate assays are shown.

FIG. 7 : Histogram depicting the results of flow cytometry sorting ofcolon CSCs. The percentage of HCT-116 CSCs which are ALDH positive andexpress CD44 and CD326 markers are shown for cells following treatmentwith PRP, IFN or PRP+IFN.

FIG. 8 : Histogram depicting the percentage of ALDH-positive HCT-116CSCs which express the markers CD44 or CD326 following treatment withPRP.

FIG. 9 : Representative light microscopy images showing sphere formationof pancreatic BxPC3 CSCs at days 2 and 5 following treatment with PRP,INF or PRP+IFN.

FIG. 10 : Representative light microscopy images of sphere formation bycolon HCT-166 CSCs at days 2 and 5 following treatment with PRP, INF orPRP+IFN.

FIG. 11 : Representative light microscopy images of neuroblastomaspheres formed when SK—N-SH cells were cultured under CSC conditions(Panel A and C). The addition of PRP to the medium prevents theformation of neuroblastoma spheres (Panel B and D). Originalmagnification: 10x for panels A and B; 20x for panels C and D. E and F:number of primary (E) and secondary (F) spheres in non-treated andPRP-treated BxPC3 and SK—N-SH cells.

FIG. 12 : Results of qRT-PCR analysis for the expression of EMT-relatedgenes after treatment with PRP (light grey). Data are normalized to 1for non-treated CSC using GAPDH as internal control, and graphed asmean±SEM (n=3).

FIG. 13 : Results of qRT-PCR analysis for the expression of CSC-relatedgenes after treatment with PRP (light grey). Data are normalized to 1for non-treated CSC using GAPDH as internal control, and graphed asmean±SEM (n=3).

FIG. 14 : A. In vivo study design for assessing the antitumor activityof PRP against CSCs. Perpendicular bars represent PRP bolus injection.B. In vivo study design for assessing the ability of PRP to inhibit theinitiation of tumour capacity of pancreatic CSCs. C. In vivo studydesign for assessing metastatic potential of CSCs in vivo. Perpendicularbars stand for PRP bolus injection.

FIG. 15 : Images of tumours excised from mice, 9.5 weeks afterinoculation with CSCs. Control group, n=10; prevention group, n=8;prevention+treatment group n=6.

FIG. 16 : Percentage tumour incidence in mice following inoculation withCSCs. Tumour incidence was measured 9.5 weeks after inoculation.Percentage incidence in prevention and prevention+treatment groups isshown as a proportion of the incidence observed in control mice (noprevention or treatment). P=0.039

FIG. 17 : Mean tumour volume (mm²) in mice ±SE, at treatment termination(9.5 weeks after inoculation). The difference in tumour weight betweenthe control group and the prevention+treatment group was statisticallysignificant, p<0.05.

FIG. 18 : A. Graph depicting the tumour volumes in each experimentalgroup (control, prevention group and prevention+treatment group),measured every 2 days following CSC inoculation. X-axis denotes thenumber of days from development of the tumour. Day 0 corresponds to 30days from CSC inoculation. B. Graph depicting the tumour volumes in eachexperimental group (control, prevention group and prevention+treatmentgroup), measured every 2 days (rate-based T/C) following CSCinoculation.

X-axis denotes the number of days from development of the tumour. Day 0corresponds to 30 days from CSC inoculation. Asterisks denotestatistical significance, p<0.05.

FIG. 19 : Tumourigenesis index (Tin) ±SE in control, prevention andprevention+treatment groups 9.5 weeks after CSC inoculation. Tin is ameasure of tumour incidence and tumour weight. The difference in Tinbetween control and both prevention and prevention+treatment groups wasstatistically significant, p_(<)0.01.

FIG. 20 : Representative images showing H&E staining. In thesubcutaneous xenograft model, tumours from mice in the control,prevention and prevention+treatment groups were excised and fixed. BxPC3tumor tissues were embedded in paraffin, sectioned with 5 pm thicknessand stained with H&E (left: magnification, 20×;middle: magnification,10×; right: magnification, x40).

FIG. 21 : Representative images showing Masson-trichome staining ofBxPC3 tumors in the subcutaneous xenograft model. Staining of tumoursfrom mice in the control, prevention and prevention+treatment groups areshown.

FIG. 22 : Expression levels of EMT-related genes in CSCs following PRPtreatment. A: expression of genes normally down-regulated during EMT butup-regulated following PRP treatment. B: expression of genes normallyup-regulated during EMT but down-regulated following PRP treatment.

FIG. 23 : The effect of PRP treatment on the expression levels of genesassociated with cell differentiation in CSCs.

FIG. 24 : The effect of PRP treatment on the expression levels of tumoursuppressor genes in CSCs.

FIG. 25 : The effect of PRP treatment on the expression levels in CSCsof genes associated with metastasis and invasion (A) and cell adhesion(B).

FIG. 26 : The effect of PRP treatment on the expression levels in CSCsof genes encoding cytokines.

FIG. 27 : The effect of PRP treatment on the expression of CSC-markergenes.

FIG. 28 : The effect of PRP treatment on the expression of MAPK-relatedgenes in CSCs.

DETAILED DESCRIPTION OF THE EMBODIMENTS

It will be understood that the invention disclosed and defined in thisspecification extends to all alternative combinations of two or more ofthe individual features mentioned or evident from the text or drawings.All of these different combinations constitute various alternativeaspects of the invention.

Reference will now be made in detail to certain embodiments of theinvention. While the invention will be described in conjunction with theembodiments, it will be understood that the intention is not to limitthe invention to those embodiments. On the contrary, the invention isintended to cover all alternatives, modifications, and equivalents,which may be included within the scope of the present invention asdefined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described. It will be understoodthat the invention disclosed and defined in this specification extendsto all alternative combinations of two or more of the individualfeatures mentioned or evident from the text or drawings. All of thesedifferent combinations constitute various alternative aspects of theinvention.

All of the patents and publications referred to herein are incorporatedby reference in their entirety.

For purposes of interpreting this specification, terms used in thesingular will also include the plural and vice versa.

Current therapeutic strategies against cancer have severe limitationsthat frequently lead to treatment failure. Accumulated evidence suggeststhat the basis for these failures is due to the inability of currenttherapies to eliminate CSCs, meaning that patients are at risk ofrecurrence and metastasis. Moreover, there is evidence to suggest theCSC populations are more resistant to conventional cancer therapies thannon-CSC populations. The elimination of CSCs is therefore critical inthe treatment of malignant diseases.

The present invention is based on the surprising finding that byproviding a combination of the pancreatic (pro)enzymes chymotrypsinogenand trypsinogen, it is possible to reduce the proliferation of CSCs,reduce the population of CSCs in a cancer cell population, inhibitsphere formation and reduce the expression of genes in CSCs associatedwith the transition from an epithelial to mesenchymal phenotype.Accordingly, the present inventors have identified a novel method fortargeting of CSCs in a cancer cell population. This finding hasimportant applications for the treatment of individuals who havepreviously received a treatment for cancer, particularly given thatconventional cancer therapies often fail to eradicate target CSCs andthere is a high likelihood of cancer recurrence, despite an apparentlysuccessful treatment of the cancer.

Accordingly, in a first aspect, the present invention provides a methodfor minimising the progression of cancer in a subject who has received atreatment for cancer, comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen.

Chymotrypsinogen (which may be abbreviated to ‘C’ herein) is a proenzymeform of the enzyme chymotrypsin, which preferentially cleaves proteinsat the following amino acids: tyrosine, tryptophan, phenylalanine andleucine. Chymotrypsin may be referred to or includes chymotrypsin A,chymotrypsin B (including B1 and B2 forms), chymotrypsin C,α-chymarophth, avazyme, chymar, chymotest, enzeon, quimar, quimotrase,α-chymar, α-chymotrypsin A, α-chymotrypsin. Chymotrypsin C can be formedfrom pig chymotrypsinogen C or from cattle subunit II ofprocarboxypeptidase A, and preferentially cleaves proteins at thefollowing amino acids: tyrosine, tryptophan, phenylalanine, leucine,methionine, glutamine, and asparagine. Chymotrypsinogen includeschymotrypsinogen A, chymotrypsinogen B1 and chymotrypsinogen B2.

Trypsinogen (which may be abbreviated to CT herein) is a proenzyme formof trypsin, which preferentially cleaves proteins at arginine andlysine. Trypsin may be referred to or include α-trypsin, μ-trypsin,cocoonase, parenzyme, parenzymol, tryptar, trypure, pseudotrypsin,tryptase, tripcellim, sperm receptor hydrolase μ-trypsin can be formedfrom trypsinogen by cleavage of one peptide bond. Further peptide bondcleavages produce a and other iso-forms. Multiple cationic and anionictrypsins can be isolated from the pancreas of many vertebrates and fromlower species including crayfish, insects (cocoonase) and microorganisms(Streptomyces griseus). In normal processes during digestion, inactivetrypsinogen is activated by enteropeptidase present in intestinal mucosato form the enzyme trypsin, which being a serine protease then acts tocleave the peptide bonds on the carboxyl side of basic aminoacids/proteins.

As described above, the proenzyme form essentially provides aninactivated form of the enzyme that becomes activated in situ (e.g. byin vivo or in vitro activation). For example, activation of theproenzyme (conversion of proenzyme to active enzyme) may occur oncontact with the CSC. It is believed that the proenzymes trypsinogen andchymotrypsinogen are selectively activated into the enzymes trypsin andchymotrypsin on contact with cancer cells and not on contact withhealthy cells. The use of proenzymes reduces problems associated withproviding, in situ, an active enzyme, such as undesirable reactions orinactivation of the enzyme before reaching an intended target of acancer cell.

Without wishing to be bound by theory, the inventors believe that thecombination of proenzymes chymotrypsinogen and trypsinogen acts toinhibit the production of growth factors and angiogenic stimulatingfactors that contribute to the tumour niche and consequently reduce thepotential for tumour engrafting. In addition, the protease proenzymesinduce the differentiation of CSCs, thereby reducing the totalpopulation of CSCs, and reducing the likelihood of recurrence ormetastasis of the tumour. For example, the inventors have shown thatchymotrypsinogen and trypsinogen, when provided to CSCs underappropriate conditions, reduce the proliferation of CSCs, reduce thepopulation of CSCs in a cancer cell population, inhibit sphere formationand reduce the expression of genes in CSCs associated with thetransition from an epithelial to mesenchymal phenotype. Given this, theuse of chymotrypsinogen and trypsinogen has particular utility inminimising the progression, recurrence or metastasis of cancer insubjects who have received a treatment for cancer, particularly giventhat conventional cancer treatments typically do not target or minimiseCSCs.

As used herein, “minimising the progression of cancer” means treatingthe subject so as to prevent or delay the recurrence or metastasis of atumour. Minimising the progression of cancer includes preventing ordelaying the recurrence of cancer, following a treatment of cancer. Therecurrence that is being prevented includes a recurrence for example, inthe tumour bed, following surgical excision. Alternatively, recurrenceincludes metastasis of the cancer in another part of the body. The terms“preventing recurrence” and “preventing relapse” as used herein, areinterchangeable.

The present invention also includes methods of preventing thedevelopment of cancer in an individual. For example the individual forwhom prevention of cancer is required may be considered to be at risk ofdeveloping cancer, but does not yet have detectable cancer. Anindividual at risk of the development of cancer may be an individualwith a family history of cancer, and/or an individual for whom genetictesting or other testing indicates a high risk or high likelihood of thedevelopment of cancer. The individual may have cancer stem cells butdoes not yet have any detectable tumours. It will be understood thatmethods of preventing the development of cancer include methods ofdelaying the onset of cancer in a subject.

The treatment previously received by the subject can be any conventionalcancer treatment, including chemotherapy, radiotherapy, immunotherapyand/or surgical excision of the tumour. In one embodiment, the treatmentreceived by the subject is surgical excision of the tumour. In anotherembodiment, for example in the treatment of non-solid tumours, thesubject has received chemotherapy, radiotherapy or immunotherapy, or acombination thereof. In yet a further embodiment, the subject hasreceived surgical, chemotherapeutic and radiotherapeutic interventionprior to being administered chymotrypsinogen and trypsinogen inaccordance with the methods of the instant invention. Any method toreduce the bulk or mass of a tumour is contemplated as a treatmentpreviously received by the subject.

The subject who has received the treatment for cancer may be in partialor complete remission. In other words, the subject, having received atreatment for cancer, as described above, may have a 50% or greaterreduction in the measurable parameters of tumour growth as may be foundon physical examination, radiologic study, or by biomarker levels from ablood or urine test. Alternatively, where the subject is in completeremission, there is a complete disappearance of all detectablemanifestations of disease, such that the subject does not have anydetectable signs of cancer. The subject may have substantiallyundetectable signs of cancer. A cancer that is “substantiallyundetectable” generally refers to a circumstance where therapy hasdepleted the size, volume or other physical measure of a cancer so thatusing relevant standard detection techniques such as in vivo imaging,the cancer, as a consequence of the therapy, is not clearly detectable.

A key limitation of conventional cancer therapies is that even aftersurgical excision of the tumour, and adjuvant therapies (such aschemotherapy), there is likely to be minimal residual disease present.Minimal residual disease (MRD) refers to the small number of cancercells that remain in a patient following treatment. Typically, thesecells are difficult to detect or cannot be detected at all, such thatthe patient is said not to have any detectable cancer. However, MRD is amajor cause of cancer relapse and the presence of CSCs even afterextensive treatment, is thought to be the key contributor to MRD.

Accordingly, the present invention provides a method of treating orpreventing minimal residual disease in a subject who has received atreatment for cancer, comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen.

The objective or outcome of treatment with chymotrypsinogen andtrypsinogen may be to reduce the number of cancer cells; reduce theprimary tumor size; inhibit (i.e., slow to some extent and preferablystop) cancer cell infiltration into peripheral organs; inhibit (i.e.,slow to some extent and preferably stop) tumor metastasis; inhibit, tosome extent, tumor growth; and/or relieve to some extent one or more ofthe symptoms associated with the disorder.

Efficacy of treatment can be measured by assessing the duration ofsurvival, time to disease progression, the response rates (RR), durationof response, and/or quality of life.

In one embodiment, the method is particularly useful for delayingdisease progression.

In one embodiment, the method is particularly useful for extendingsurvival of the subject, including overall survival as well asprogression free survival.

In one embodiment, the method is particularly useful for providing acomplete response to therapy whereby all signs of cancer in response totreatment have disappeared. This does not always mean the cancer hasbeen cured.

In one embodiment, the method is particularly useful for providing apartial response to therapy whereby there has been a decrease in thesize of one or more tumors or lesions, or in the extent of cancer in thebody, in response to treatment.

The methods of the present invention also include targeted treatment ofCSCs before a subject receives a conventional treatment for cancer. Theskilled person will appreciate that this approach has particularapplication for haematologic cancers, where it is not possible tosurgically excise a tumour. As such, the invention provides a method ofsensitising a subject prior to treatment for cancer, the methodcomprising administering a therapeutically effective amount ofchymotrypsinogen and trypsinogen to the subject before the subjectreceives treatment for cancer.

Still further, the methods of the present invention also includetargeted treatment of CSCs at the same time as a subject receives aconventional treatment for cancer. As such, the invention provides amethod of further treating a subject for cancer, including preventingthe likelihood of minimal residual disease at the completion oftreatment or delaying or preventing the recurrence of cancer, the methodcomprising administering a therapeutically effective amount ofchymotrypsinogen and trypsinogen to the subject concurrently with theconventional treatment for cancer.

Identification of cancer stem cells may be by detection of one or moremarkers. Exemplary markers are shown Table 1 below.

Tumor type Phenotype of CSCs markers LeukemiaCD34⁺CD38⁻HLA-DR-CD71⁻CD90⁻CD117⁻CD123⁺ Breast cancerESA⁺CD44⁺CD24^(−/low)Lineage⁻, ALDH-1^(high) Liver cancer CD133⁺,CD49f⁺, CD90⁺ Brain cancer CD133⁺, BCRP1⁺, A2B5⁺, SSEA-1⁺ Lung cancerCD133⁺, ABCG2^(high) Colon cancer CD133⁺, CD44⁺, CD166⁺, EpCAM⁺, CD24⁺or CD326⁺ and CD44⁺ Multiple myeloma CD138⁻ Prostate cancer CD44⁺,α2β1^(high), CD133⁺ Pancreatic CD133⁺, CD44⁺, EpCAM⁺, CD24⁺ or CD326⁺,CD44⁺ and CxCR4⁺ Melanoma CD20⁺ Head and neck CD44⁺ cancer

Cancer stem cells may be isolated from a tissue or sample using anymethod described herein. Cancer stem cells from a sample of the tumouror tumour stroma may be identified by sectioning the at least part ofthe sample, labelled (preferably immunolabelled) for any one or morecancer stem cells markers and analysed by immunofluorescence.

The “subject” includes a mammal. The mammal may be a human, or may be adomestic, zoo, or companion animal. While it is particularlycontemplated that the methods of the invention are suitable for medicaltreatment of humans, they are also applicable to veterinary treatment,including treatment of companion animals such as dogs and cats, anddomestic animals such as horses, cattle and sheep, or zoo animals suchas felids, canids, bovids, and ungulates. A subject may be afflictedwith cancer or other disorder, or may not be afflicted with cancer orother disorder (i.e., free of detectable disease).

The typical body weight of a human subject may be greater than, or equalto, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 or 110 kg.

The term “therapeutically effective amount” refers to an amount ofcomposition, or agent or compound in the composition, capable ofminimising the progression of, treating, preventing recurrence of orameliorating cancer or the spread (metastasis) thereof. Atherapeutically effective amount may be determined empirically and in aroutine manner in relation to treating cancer, and will result inincreased life expectancy.

As described herein, methods of the invention include minimising theprogression, preventing the recurrence of or treating minimal residualdisease associated with neoplasms and related conditions, cancers,tumours, malignant and metastatic conditions. Tissues and organsassociated with solid tumours and metastases which can be treated with amethod or pharmaceutical composition of the invention include, but arenot limited to, biliary tract, bladder, blood, brain, breast, cervix,colon, endometrium, oesophagus, head, neck, kidney, larynx, liver, lung,medulla, melanin, ovarian, pancreas, prostate, rectum, renal, retina,skin, stomach, testes, thyroid, urinary tract, and uterus.

The methods and pharmaceutical compositions of the invention are usefulfor minimising the progression including preventing recurrence ofcancers and metastatic carcinomas of the following types: pancreaticcancer, oesophageal cancer, colon cancer, bowel cancer, prostate cancer,ovarian cancer, stomach cancer, breast cancer, malignant melanoma,neuroblastoma or lung cancer. Preferably, the cancer is pancreaticcancer, colon cancer or ovarian cancer. More preferably, the cancer ispancreatic cancer.

The methods and pharmaceutical compositions of the invention may providea multiple effect approach to treating cancer, for example by increasingapoptosis in tumour cells, increasing cell-to-cell adhesion,differentiation and immunogenicity (targeting and removal by immunesystem). It is therefore beneficial to conduct treatment in the absenceof any other treatments that may suppress or harm the immune system.

In addition to providing methods of treatment, it will be appreciatedthat the instant invention includes the use of therapeutically effectiveamounts of chymotrypsinogen and trypsinogen in the manufacture ofmedicament for minimising the progression of cancer in a subject who hasreceived a treatment for cancer.

In further embodiments, the use includes use of chymotrypsinogen andtrypsinogen in the manufacture of a medicament for treating minimalresidual disease in a subject, or for sensitising a subject before theyreceive a treatment for cancer.

The methods of the instant invention involve the administration ofchymotrypsinogen and trypsinogen to a subject in need thereof.

The trypsinogen and chymotrypsinogen used in any aspect of the inventionmay be isolated, purified, substantially purified, recombinant orsynthetic.

The proenzymes trypsinogen and chymotrypsinogen may be precursors of theenzymes selected from chymotrypsin classes 3.4.21.1 or 3.4.21.2 ortrypsin from class 3.4.21.4, or selected from any other suitable source(classes grouped according to the classification of the NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology). These enzymes are commercially available and may be of human,bovine or porcine origin.

In certain aspects of the invention, the chymotrypsinogen andtrypsinogen may be administered simultaneously or sequentially. Whenadministered simultaneously, the chymotrypsinogen and trypsinogen can beincluded in the same pharmaceutical composition. When combined in thesame pharmaceutical formulation, the weight ratio ofchymotrypsinogen:trypsinogen may be in the range of between 1: 1 to ator about 10 : 1, at or about 4: 1 to at or about 8: 1, at or about 5: 1to at or about 7: 1, or at about 6: 1. In one embodiment, the weightratio of chymotrypsinogen:trypsinogen is in the range of between 5:1 to7:1, preferably 6:1.

In one aspect, the pharmaceutical formulations of the instant inventioncomprise only chymotrypsinogen and trypsinogen as active agents. Inalternative embodiments, additional active agents are included in thecomposition. For example, in addition to the pancreatic proenzymeschymotrypsinogen and trypsinogen, the compositions may include otherknown therapies for cancer treatment.

The pharmaceutical compositions of the invention may be formulated, forexample, by employing conventional solid or liquid vehicles or diluents,as well as pharmaceutical additives of a type appropriate to the mode ofdesired administration (for example, excipients, binders, preservatives,stabilizers, flavours, etc.) according to techniques such as those wellknown in the art of pharmaceutical formulation.

The pharmaceutical compositions of the invention, and preparations orformulations thereof may be prepared by admixing together the componentsof the composition, including chymotrypsinogen and trypsinogen. Theadmixing may be performed sequentially or simultaneously.

The pharmaceutical compositions of the invention may conveniently bepresented in dosage unit form and may be prepared by any of the methodswell known in the art of pharmacy. All methods include the step ofbringing the active agents and/or protease proenzyme into associationwith the carrier which constitutes one or more accessory ingredients. Ingeneral, the pharmaceutical compositions are prepared by uniformly andintimately bringing the active agents and/or protease proenzymes intoassociation with a liquid carrier or a finely divided solid carrier orboth, and then, if necessary, shaping the product into the desiredformulation. The active agents and/or protease proenzymes are providedin a dosage unit form in an amount sufficient to produce the desiredeffect upon the process or condition of diseases after single orrepeated administration.

The pharmaceutical compositions of the invention may be in a formsuitable for oral use, for example, as tablets, troches, lozenges,aqueous or oily suspensions, dispersible powders or granules, emulsions,hard or soft capsules, or syrups or elixirs. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavouring agents, colouring agents and preservingagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the protease proenzyme and active agent ofthe first and second aspects in admixture with non-toxicpharmaceutically acceptable excipients which are suitable for themanufacture of tablets. These excipients may be for example, inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets may be uncoated or they maybe coated by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated to form osmotic therapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the protease proenzyme and active agent of the first and secondaspects are mixed with an inert solid diluent, for example, calciumcarbonate, calcium phosphate or kaolin, or as soft gelatin capsuleswherein the protease proenzyme and active agent of the first and secondaspects are mixed with water or an oil medium, for example peanut oil,liquid paraffin, or olive oil.

Aqueous suspensions contain the active agent and protease proenzyme inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, μ-hydroxybenzoate, one or more colouringagents, one or more flavouring agents, and one or more sweeteningagents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active agent andprotease proenzyme in a vegetable oil, for example arachis oil, oliveoil, sesame oil or coconut oil, or in a mineral oil such as liquidparaffin. The oily suspensions may contain a thickening agent, forexample beeswax, hard paraffin or cetyl alcohol. Sweetening agents suchas those set forth above, and flavouring agents may be added to providea palatable oral preparation. These may be preserved by the addition ofan anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the protease proenzyme andactive agent of the first and second aspects in admixture with adispersing or wetting agent, suspending agent and one or morepreservatives. Suitable dispersing or wetting agents and suspendingagents are exemplified by those already mentioned above. Additionalexcipients, for example sweetening, flavouring and colouring agents, mayalso be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally- occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweeteningand flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. They may also contain ademulcent, a preservative and flavouring and colouring agents.

The pharmaceutical compositions of invention may be in the form of asterile injectable aqueous or oleagenous suspension. This suspension maybe formulated according to the known art using those suitable dispersingor wetting agents and suspending agents which have been mentioned above.The pharmaceutical compositions of the first and second aspects may alsobe a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butane diol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

In a particular embodiment, the pharmaceutical compositions of theinvention are formulated as suppositories for rectal administration ofthe drug. These formulations can be prepared by mixing the proteaseproenzyme and active agent of the first and second aspects with asuitable non-irritating excipient which is solid at ordinarytemperatures but liquid at the rectal temperature and will thereforemelt in the rectum to release the drug. Such materials include cocoabutter and polyethylene glycols. Rectal administration may be used toeliminate entero-hepatic first pass effect in the gastro-intestinaltract related to oral administration of enzymes.

The pharmaceutical compositions of the invention, may also be formulatedin liposomes. As is known in the art, liposomes are generally derivedfrom phospholipids or other lipid substances. Liposomes are formed bymono- or multilamellar hydrated liquid crystals that are dispersed in anaqueous medium. Any non-toxic, physiologically acceptable andmetabolisable lipid capable of forming liposomes can be used. Theliposome formulation may contain stabilisers, preservatives, excipientsand the like. The preferred lipids are the phospholipids andphosphatidyl cholines, both natural and synthetic. Methods to formliposomes are known in the art.

The pharmaceutical compositions of the invention, may be included in acontainer, pack, or dispenser together with instructions foradministration. The protease proenzymes and active agents, andoptionally additional active agent, of the pharmaceutical compositionmay be provided as separated components in the container, pack, ordispenser, to be taken separately or together at the same or differenttime in a use or method of the invention described herein.

An appropriate dosage level for the pharmaceutical compositions of theinvention will generally be about 0.01 to 500 mg per kg patient bodyweight per day which can be administered in single or multiple doses. Itmay be about 0.1 to about 250 mg/kg per day; or about 0.5 to about 100mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kgper day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg perday. Within this range the dosage may be 0.05-0.5, 0.5-5 or 5-50 mg/kgper day.

For oral administration, the pharmaceutical compositions may be providedin the form of tablets containing 1.0-4000 milligrams of the proteaseproenzyme particularly 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0,100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 750.0, 1000, 1500,2000, 2500, 3000, 3500, and 4000 milligrams of the protease proenzymesfor the symptomatic adjustment of the dosage to the patient to betreated. The pharmaceutical compositions as described herein may beadministered on a regimen of 1 to 4 times per day, once or twice perday, or once daily, with reduced requirements of administrationgenerally leading to greater compliance.

The dosage may vary widely depending on whether a single administrationform of the composition is given. A suitable single administration foran embodiment of the pharmaceutical compositions as described herein maycomprise:

-   -   Trypsinogen in an amount of between 1-100 mg, particularly 2-50        mg, more particularly (in mg) 1.0, 2.5, 5.0, 7.5, 10.0, 15.0.        20.0, 25.0, 30.0, 40.0, 50.0; and    -   Chymotrypsinogen in an amount of between 1-100 mg, particularly        2-50 mg, more particularly (in mg) 1.0, 2.5, 5.0, 7.5, 10.0,        15.0. 20.0, 25.0, 30.0, 40.0,50.0.

It will be understood, however, that the specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the host undergoing therapy.

Suitable dosage levels for the various components (if present) of anembodiment of the pharmaceutical compositions as described herein, maycomprise:

-   -   Chymotrypsinogen in an amount of at least 0.2 mg/kg, or in a        range of 0.2-5 mg/kg, in a range of 0.5-2.0 mg/kg, or about 0.8        mg/kg;    -   Trypsinogen in an amount of less than 0.5 mg/kg, or in a range        of 0.01-0.4 mg/kg, or in a range of 0.05-0.20 mg/kg, or about        0.1 mg/kg;

Suitable concentrations for the various components (if present) of thepharmaceutical compositions as described herein, which may beparticularly effective if present at or near the surface of a tumourcell, may comprise:

-   -   Chymotrypsinogen in a concentration of at least 0.5 mg/ml, or in        a range of 1-2 mg/ml;    -   Trypsinogen in a concentration of less than 0.25 mg/ml, or in a        range of 0.1-0.2 mg/ml;

The pharmaceutical compositions of the invention may be administered byany suitable means, for example, orally, such as in the form of tablets,capsules, granules or powders; sublingually; buccally; parenterally,such as by subcutaneous, intravenous, intramuscular, or intracisternalinjection or infusion techniques (e.g., as sterile injectable aqueous ornon-aqueous solutions or suspensions); nasally such as by inhalationspray; topically, such as in the form of a cream or ointment; orrectally such as in the form of suppositories; in dosage unitformulations containing non-toxic, pharmaceutically acceptable vehiclesor diluents. They may, for example, be administered in a form suitablefor immediate release or extended release, for example, by the use ofdevices such as subcutaneous implants, encapsulated spheroids or osmoticpumps.

In certain embodiments, it may be preferably to administer thechymotrypsinogen and trypsinogen directly to the tumour bed orsurrounding tissues, for example following excision of the tumour mass.In alternative embodiments, the chymotrypsinogen and trypsinogen areprovided as a systemic dose, which may be more suitable for haematologiccancers.

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly, and are not intended to limit the scope of the invention.

EXAMPLES Example 1

A series of in vitro studies were performed to study the effect ofpancreatic pro-enzymes on proliferation and differentiation of CSCs.

A. Materials and Methods

Treatment Solutions and Control

A stock solution of 6 mg/ml of Chymotrypsinogen A and 1 mg/ml ofTrypsinogen was prepared in PBS and stored at −20° C. until required.For each experiment, the stock solution was further diluted in culturemedium to obtain the desired concentrations (see Table 2).

The anti-proliferative agent Interferon alpha (IFN) was used as apositive control at a concentration of 10000 UI/ml (INTRON® A Interferonalfa-2b).

A solution comprising Chymotrypsinogen A/Trypsinogen and IFN was alsoused to assess the combined effect of these reagents.

Cell Lines

The human pancreatic cancer cell lines BxPC3 and the human colon cancercell line HCT-116 were obtained from American Type Culture Collection(ATCC) and maintained in RPM! 1640 Medium (for BxPC3) and Dulbecco'sModified Eagle Medium (for HCT-116) (Sigma-Aldrich) supplemented with10% FBS.

The neuroblastoma cell line SK—N-SH (ATCC® HTB-11™ derived fromneuroblastoma at metastatic site, bone marrow), was obtained fromAmerican Type

Culture Collection (ATCC) and maintained in Eagle's Minimum EssentialMedium supplemented with 10% FBS.

Cell passaging was performed using the trypsin replacement reagentTrypLE™ (Life Technologies).

Isolation of CSCs

BxPC3 pancreatic and HCT-116 colon cancer cells lines were grown insphere forming medium in ultralow attachment plates. After 1 week, cellsformed spheres and cancer stem-like cells were isolated using theALDEFLUOR assay (StemCell Technologies) and the following markers:CD326, CD44 and CxCR4 (BxPC3) and CD326 and CD44 (for HCT-116) byfluorescence-activated cell sorting (FACS). The enriched subpopulationof CSCs were grown with a specific sphere-forming medium in ultralowattachment plates (Corning) as previously described in Charafe-Jauffretet al. (2010) Clin Cancer Res.16(1):45-55. A similar method is describedin Ramirez et al. (2014) Oncotarget, 5(11): 3590-3606.

Determination of Optimal Concentration of Trypsinogen/Chymotrypsinogento Use in Downstream Assays

Cell viability and proliferation was assessed using the MTT(3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay(Sigma). Briefly, cells (2×10³ cells/well) were seeded onto 96-wellplates and incubated for 24 h and then treated with different solutionsof Trypsinogen/Chymotrypsinogen, (see Table 2), IFN (control) andTrypsinogen/Chymotrypsinogen+IFN.

Three days later, treatment was repeated and cells were maintained for 3additional days. Cells were maintained with the treatment for six days.Thereafter, cells were processed as follow, 10 pL of 2 mM MTT reagentwas added to each well and cells were incubated at 37° C. for 4 hours.100 pL detergent reagent was then added and cells were leaved at roomtemperature in the dark for 2 hours. Absorbance was recorded at 570 nm.

The IC₅₀ values were calculated from four parametric logistic curves bylinear interpolation using Sigma Plot software. All of the experimentswere plated in triplicate wells and were carried out at least twice.

The effect of anticancer drugs on cell viability and proliferation wasassessed using the MTT (3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay (Sigma).

TABLE 2 Composition and concentration of treatment solutions Treatmentsolution composition (mg/ml) Reagent 1 2 3 4 5 6 Chymotrypsinogen 0.030.06 0.18 0.3 0.42 0.6 Trypsinogen 0.005 0.01 0.03 0.05 0.07 0.1

Cell Cycle Assay

Cell cycle distribution was measured in control and treated cells. Aftertreatment, cells were collected in 15 ml tubes and washed once in cold 5ml PBS. To fix the cells, cells were vortexed gently while 200 μl of 70%ethanol was added drop-by-drop and incubated at least for 20 min at 4°C. in the dark. After fixation the ethanol was discarded bycentrifugation and cells were stained by addition of a solutioncontaining PI and RNase and incubated in the dark for 45 minutes. Thesamples were immediately processed using a FACSAria III flow cytometer(Becton Dickinson, BD Biosciences, Franklin Lakes, N.J., USA) from theScientific Instrumental Center (University of Granada). At least 10,000events were collected in each final gated histogram. Cell cycle analysiswas performed using Dean and Jett's algorithm (Multicycle, Phoenix FlowSystems, San Diego, Calif.).

CSC Marker Analysis by Flow Cytometry

ALDEFLUOR sorted BxPC3 pancreas and HCT-116 colon cancer cells weremaintained for 6 days in culture and treated twice (day 2 and day 5)with Chymotrypsinogen: 0.42 mg/ml - Trypsinogen: 0.07 mg/ml.

Treated and untreated CSCs were disassociated using Tryple and washedtwice in PBS supplemented with 1% bovine serum albumin (Sigma-Aldrich,St Louis, Mo.). The cell surface Fc receptor was blocked using IgG(Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.) on ice for 15 min.Cells were stained for 30 min at 4° C. with anti-CD44-PE and anti-CD326FITC and CxCR4-APC monoclonal antibodies (BD Biosciences, FranklinLakes, N.J.). After washing, cells were analysed using a FACSAria IIIflow cytometer (Becton Dickinson, BD Biosciences, Franklin Lakes, N.J.,USA) from the Scientific Instrumental Center (University of Granada).

CSC Sphere Formation in the Presence of Trypsinogen/Chymotrypsinogen

SK—N—SH cells were detached from the culture flask and cultured inCSC-enriched conditioned medium in presence ofTrypsinogen/Chymotrypsinogen (0.07:0.42 mg/ml). Sphere formation wasmonitored every day under light microscopy, pictures were taken on day6.

BxPC3 pancreatic and HCT-116 colon cancer cells lines were detached fromthe culture flask and cultured in CSC-enriched conditioned medium inpresence of either:

1. Trypsinogen/Chymotrypsinogen (0.07 mg/ml:0.42 mg/ml)

2. Trypsinogen/Chymotrypsinogen (0.07 mg/ml:0.42 mg/ml) plus IFN (10000UI/ml); or

3. IFN (10000 UI/ml)

Sphere formation was monitored every day under light microscopy,pictures were taken on day 2 and day 5.

For the secondary sphere-forming assay, cells from primary spheres werecollected by centrifugation, then dissociated with Tryple andmechanically disrupted with a pipette. 10³ single cells were plated andresuspended in spheres culture medium in ultra-low adherence 24-wellplates. Spheres >75 p M diameter were counted after 6 days by lightmicroscopy.

Quantitative Real Time RT-PCR

Total RNA from the different cell lines was extracted from duplicate 80%confluent cultures using the TRIZOL reagent following the instructionsof the manufacturer (Life Technologies). cDNA was synthesized by reversetranscription of total RNA using the Reverse Transcription System(Promega) and qRT-PCR assay was done using SYBR Green PCR Master Mix(Promega) and random primers. Each reaction was performed in triplicatefrom two cDNA dilutions. The comparative threshold cycle (Ct) method wasused to calculate the amplification factor as specified by themanufacturer. Human GADPH was used as an internal standard to normalizevariations in RNA quality in the quantities of input cDNA. Primersequences for determining expression of genes associated withEpithelial-mesenchymal transition (EMT) are listed Table 3.

TABLE 3 Primer sequences used to determine expression levels of genesassociated with Epithelial- mesenchymal transition (EMT) GenePrimer Sequence SNAIL Forward 5′ ACCCCACATCCTTCTCACTG 3′ (SEQ ID NO: 1)Reverse 5′ TACAAAAACCCACGCAGACA 3′ (SEQ ID NO: 2) SLUG Forward5′ TGCGATGCCCAGTCTAGAAA 3′ (SEQ ID NO: 3) Reverse5′ TTCTCCCCCGTGTGAGTTC 3′ (SEQ ID NO: 4) E-CADHERIN Forward5′ AATTCCTGCCATTCTGGGGA 3′ (SEQ ID NO: 5) Reverse5′ TCTTCTCCGCCTCCTTCTTC 3′ (SEQ ID NO: 6) N-CADHERIN Forward5′ TGAGCCTGAAGCCAACCTTA 3′ (SEQ ID NO: 7) Reverse5′ AGGTCCCCTGGAGTTTTCTG 3′ (SEQ ID NO: 8) VIMENTIN Forward5′ AGCTAACCAACGACAAAGCC 3′ (SEQ ID NO: 9) Reverse5′ TCCACTTTGCGTTCAAGGTC 3′ (SEQ ID NO: 10) OCT4 Forward5′ CACCATCTGTCGCTTCGAGG 3′ (SEQ ID NO: 11) Reverse5′ AGGGTCTCCGATTTGCATATCT 3′ (SEQ ID NO: 12)

TABLE 4 Primer sequences used to determine expression levels of genesassociated with pluripotency Gene Primer Sequence KLF4 Forward5′ CGAACCCACACAGGTGAGAA 3′ (SEQ ID NO: 13) Reverse5′ TACGGTAGTGCCTGGTCAGTTC 3′ (SEQ ID NO: 14) SOX 2 Forward5′ CAAGATGCACAACTCGCAGA 3′ (SEQ ID NO: 15) Reverse5′ CATGAGCGTCTTGGTTTTTCC 3′ (SEQ ID NO: 16) NANOG Forward5′ TCCTGAACCTCAGCTACAAAC 3′ (SEQ ID NO: 17) Reverse5′ GCGTCACACCATTGCTATTC 3′ (SEQ ID NO: 18) C-MYC Forward5′ GAGTCTGGATCACCTTCTGCTG 3′ (SEQ ID NO: 19) Reverse5′ AGGATAGTCCTTCCGAGTGGAG 3′ (SEQ ID NO: 20) CD133 Forward5′ CAGAATAATAAACAGCAGCCC 3′ (SEQ ID NO: 21) Reverse5′ GATTATGACAAGCCAGAAACT 3′ (SEQ ID NO: 22)

Microarray Studies

The expression of genes involved in epithelial to mesenchymal transition(EMT) or related to CSC expression was analysed with RT² Profiler™ PCRArrays (Qiagen) processed following manufacturer's instructions.

A set of controls present on each array enabled data analysis using theAACT method of relative quantification and assessment of reversetranscription performance, genomic DNA contamination, and PCRperformance.

The RT² Profiler™ PCR Arrays used were:

1. The Human Epithelial to Mesenchymal Transition (EMT) RT² Profiler™PCR Array, that profiles the expression of 84 key genes that eitherchange their expression during EMT or regulate gene expression changesduring EMT. The array includes cell surface receptor, extracellularmatrix, and cytoskeletal genes mediating cell adhesion, migration,motility, and morphogenesis; genes controlling cell differentiation,development, growth, and proliferation; as well as signal transductionand transcription factor genes that cause EMT and all of its associatedprocesses.

2. The Human Cancer Stem Cells RT² Profiler PCR array profiles theexpression of 84 genes linked to cancer stem cells (CSCs). The genesprofiled with this array include CSC molecular markers and genesregulating CSC proliferation, self-renewal, and pluripotency to helpensure the stability of CSC isolates in culture. Also included are genesinvolved in CSC asymmetric cell division, migration and metastasis, andrelevant signal transduction pathways.

B. Results

IC₅₀ Determination by MTT Assay

The MTT assay is based on the reduction of MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) bymetabolically active cells. The resulting intracellular purple formazancan be solubilized and quantified by spectrophotometric means.

FIG. 1 shows the response curves at various concentrations ofTrypsinogen/Chymotrypsinogen and Trypsinogen/Chymotrypsinogen+IFN.

The data show that the optimal concentration ofTrypsinogen/Chymotrypsinogen A is Trypsinogen 0.07 mg/ml andChymotrypsinogen A 0.42 mg/ml (a 1:6 ratio), however ratios of between1:1 to about 1:10 Trypsinogen/Chymotrypsinogen are also expected toreduce cell metabolic activity and inhibit cell proliferation. Theseconcentrations of Trypsinogen 0.07 mg/ml and Chymotrypsinogen A 0.42mg/ml were used in subsequent experiments and is referred to as “PRP” insubsequent experiments. The data also show that the combination of IFNand Trypsinogen/Chymotrypsinogen significantly enhances theantiproliferative effect of Trypsinogen/Chymotrypsinogen.

Cell Cycle Assay

Cell cycle assays were performed on cells that were grown under sphereformation conditions (CSCs) and on adherent cells (non-CSCs). Cells weretreated once with IFN (10000 Wimp, PRP (Trypsinogen/Chymotrypsinogen0.07 mg/ml:0.42 mg/ml) and PRP+IFN (Trypsinogen/Chymotrypsinogen 0.07mg/ml:0.42 mg/ml plus IFN 10000 Ul/ml) and after 4 days processed forcell cycle assay.

The numbers of cells in G1/G0 phase were similar in pancreatic CSCs andnon-CSCs (72.8% vs 82.4%) and in colon CSCs and non-CSCs (77.1% vs79.9%). Treatment with PRP did not appear to change the number of cellsin G1/GO in pancreatic CSC (82.4% vs 89.4%) or in colon CSC (79.9% vs78.8%) (FIG. 2 a-c ; FIG. 3 a-c ).

PRP treatment resulted in a slight decrease in the number of cells inphase S compared with the control (3.25% vs 7.95%), an effect that waspotentiated with the addition of IFN (2.25%) (FIG. 2 d ). There was nomarked difference in cell cycle in colon CSCs after treatment with PRP(FIG. 3 d ).

These results suggest that Trypsinogen/Chymotrypsinogen decreases theability of pancreatic CSCs to proliferate and reduces S phase inpancreatic CSCs.

ALDEFLUOR Assay

CSC mechanisms for survival include an increased metabolic activitythrough aldehyde dehydrogenase (ALDH). For this reason ALDH has beendescribed as a marker for the identification of cancer stem cells (Huanget al. (2009). Cancer Res Vol.69 (No. 8):3382-3389). ALDH positive cellscan be detected with ALDEFLUOR reagent by using flow cytometry.Aldefluor assay is based on the conversion of fluorescent non-toxicsubstrate for ALDH substrate to the fluorescent reaction product.Non-toxic substrate for ALDH can freely diffuse into intact, viablecells. The BODIPY aminoacetaldehyde is converted to the fluorescentproduct BODIPY aminoacetate by ALDH activity. ALDH positive cells havebeen found in various cancer tissues including breast, liver, colon,pancreas, prostate, lung, ovarian and acute myelogenous leukemia and arerelated to cancer chemo resistance (Siclari and Qin (2010) J Orthop SurgRes 5(78)).

FIG. 4 shows the results of ALDH assay on pancreatic CSCs and non-CSCs.

The percentage of ALDH-positive cells in non-CSC samples wasapproximately 12%. For cells cultured under sphere-forming conditions,approximately 50% of cells were ALDH-positive. Treatment of cells withPRP significantly reduced the population of ALDH-positive cells (from47.2% to 17.5%) in pancreatic CSCs. The addition of IFN furtherincreased the effect of PRP (13.8%). These results indicate thatTrypsinogen/Chymotrypsinogen reduces the CSC population in pancreaticcancer.

Expression of CSC Markers in Trypsinogen/Chymotrypsinogen-Treated Cells

Pancreatic cancer cells were grown under sphere-forming conditions toenrich the subpopulation of CSCs. Pancreatic CSCs were then treated withPRP (Trypsinogen/Chymotrypsinogen 0.07 mg/ml:0.42 mg/ml), PRP+IFN andIFN and the pancreatic-specific CSC markers CD 326, CD 44 and CxCR4 weremeasured by flow cytometry.

Flow cytometry results demonstrate that the percentage of cellsexpressing the three CSC markers significantly decreases after treatmentwith PRP (from 56.6% to 8.8%). Treatment with PRP+IFN reduced thepopulation of cells expressing those markers to 4.3% (FIGS. 5 and 6 ).These results suggest that treatment with PRP reduces the CSC populationin pancreatic cancer.

Colon cancer cells were grown under sphere formation condition to enrichthe subpopulation of CSCs. Colon CSCs were then treated with PRP,PRP+IFN and IFN. Cells were marked with ALDH together with thecolon-specific CSC markers: CD326 and CD44 and analysed by flowcytometry.

Flow cytometry results (FIGS. 7 and 8 ) demonstrate that the percentageof cells that were both ALDH-positive and expressed both colon-specificCSC markers significantly decreased after PRP treatment (from 64.8% to31.05%). Treatment with PRP+INF reduced the population of cells thatexpressed those markers to 40.8% (FIG. 7 ). The percentage of cells thatwere ALDH+ and express either CD44 or CD326 markers significantlydecreased after PRP treatment (from 35.5% to 8.1% for ALDH+CD44+ cellsand from 43.9 to 7.1% for ALDH+CD326+ cells— FIG. 8 ).

CSC Sphere Formation in Presence of Trypsinogen/Chymotrypsinogen

The ability of PRP to suppression sphere formation was tested. PRP(Trypsinogen/Chymotrypsinogen 0.07 mg/ml:0.42 mg/ml), IFN (10000 UI/ml)and PRP+IFN were added to the medium at day 0, when the cancer cellswere cultured under sphere conditions (conditioned medium and lowattachment culture wells).

FIG. 9 show images of the pancreatic cell cultures on day 2 and day 6.The day 2 images show that the addition of PRP to the mediumsignificantly reduces the number and size of pancreatic CSC spheres. Inaddition, the morphology of the spheres formed is markedly different(being regular in the control non-treated cells while irregular inPRP-treated cells).

The day 5 images show key differences betweenTrypsinogen/Chymotrypsinogen A-treated cells and control cells. PRP andPRP+IFN treated cells do not form spheres. Cells appear to bedisassociated and ruptured.

The results with the colon cancer cells were different. FIG. 10 showthat the addition of PRP inhibited the formation of the spheres even atday 2. Cells appear disperse but not dead. On day 5 the sphere had notyet been formed and the cells were not aggregated.

These results indicate that Trypsinogen/Chymotrypsinogen markedlyinhibits spheroid formation by CSCs.

Neuroblastoma CSC Sphere Formation in Presence of PRP

PRP (Trypsinogen/Chymotrypsinogen 0.07 mg/ml:0.42 mg/ml), was added tothe cell medium at day 2 and at day 4, when the cancer cells werecultured under sphere conditions (conditioned medium and low attachedculture wells).

FIG. 11 shows images of the cell cultures on day 6. Neuroblastomatreated with PRP do not form CSC spheres: cells grow in suspension butdo not form cell clusters (FIG. 11 B and D). Neuroblastoma sphere CSCsare shown in panels A and C, when cells were maintained under controlconditions in the absence of PRP.

Secondary Sphere Formation

A secondary sphere-formation assay was performed by dissociating primaryspheres formed by PRP treated cells and comparing with controlnon-treated cells. FIG. 11E shows that for the cancer cell lines BxPC3and SK—N—SH, no secondary spheres could be counted after PRP treatment.In contrast non-treated control cells were able to recover the abilityto form cell clusters.

PRP destroys primary spheres and also suppresses the ability of CSCs toform secondary spheres. As tumor spheres are defined as clonally derivednon-adherent colonies of cells derived from a single tumor stem cell,the results obtained demonstrate that PRP treatment has a direct effecton the cancer stem cell population.

EMT and Pluripotency-Associated Gene Expression

The expression of genes related with the EMT process and/or pluripotencywas analysed in pancreatic CSCs treated with PRP(Trypsinogen/Chymotrypsinogen A 0.07 mg/ml:0.42 mg/ml), and comparedwith untreated CSCs. qRT-PCR results show that genes related to EMT suchas SNAIL, SLUG, VIMENTIN and N-CADHERIN had decreased expression aftertreatment (FIG. 12 ). E-CADHERIN expression was increased aftertreatment. These results suggest that PRP treatment may reduce theconversion from an epithelial to a mesenchymal phenotype.

To determine if PRP treatment alters expression of CSC markers, severalgenes of pluripotency were analysed by qRT-PCR (primers used aredescribed in Table 4). The results show that OCT4, CMYC and KLF4 geneexpression slightly decreased following treatment withTrypsinogen/Chymotrypsinogen A when compared with non-treated CSCs. Incontrast, expression of NANOG and CD133 increased after treatment,indicating that the effect of PRP is not mediated though these two genes(FIG. 13 ).

Microarray Studies

The following results relate to changes in gene expression in CSCsfollowing PRP treatment.

a) Expression Genes Associated with EMT

To study the effect of PRP on EMT process, the expression of several keygenes involved in EMT was analysed in treated CSCs and compared with thegene expression profile of control non-treated CSCs. Results are shownas the fold regulation (up or down) compared with control cells.

FIG. 22A shows the change in gene expression of select genes that arenormally down regulated during the EMT process. FIG. 22A shows thatfollowing PRP treatment, expression of E-cadherin increased 4.28-fold,indicating that PRP treatment induces expression of this gene.Furthermore, Kruppel-like-factor 17 (KLF17) was also up-regulated by PRPtreatment, reaching a fold up-regulation of 5.9. KLF17 has previouslybeen reported to directly supress EMT, angiogenesis, invasion andmetastasis. The expression of other genes was also increased followingPRP treatment (e.g., MST1R: 1.6928-fold; TFPI2: 4.4272-fold and NUDT13:2.3692-fold), suggesting that PRP has an anti-EMT effect.

Genes that are normally up regulated during EMT were analysed and theresults are summarized in FIG. 22B. 13 genes that have been described toinduce EMT were down regulated following treatment with PRP. Forexample, the transcription factor Snail, which is typically up-regulatedin metastatic cells that are undergoing EMT, was down regulatedfollowing PRP treatment (SNAI1:-1.9678 and SNAI2:-1.8604). The serineprotease inhibitor SERPINE1, was significantly down-regulated (4.6-folddownregulation) following PRP treatment. Expression of the integrinsITGA5 and ITGAV was also was down regulated (2.5697 and 1.0193downregulation, respectively). ITGA5 has been reported to promote tumorinvasion and higher expression of this gene has previously beencorrelated with a shorter survival time in cancer patients. ITGAV isalso implicated in the regulation of angiogenesis and cancerprogression.

In conclusion, PRP treatment has a significant effect on the expressionof genes that are involved in the EMT process, by inducing theexpression of genes normally downregulated during EMT, and reducing theexpression of genes normally upregulated during EMT. The resultsindicate the therapeutic potential for PRP in reducing tumor progressionand metastasis, in addition to repressing the CSC population.

b) Expression of Genes Involved in Stem Cell Differentiation

The expression of key genes involved in pancreatic differentiation wereanalysed in pancreatic CSCs. The expression of genes in CSCs treatedwith PRP was compared with expression of genes in control non-treatedCSCs. Results are showed as the fold regulation (up or down) comparedwith control cells.

The results obtained are summarized in FIG. 23 , and were concordantwith studies, showing that PRP treatment induces cell differentiation. 7genes related to differentiation were up regulated following PRPtreatment, including the expression of epidermal growth factor (EGF),which increased 8.9 fold when compared with control cells.

c) Expression of Tumor Suppressor Genes

FIG. 24 shows the up-regulation of 11 genes with previously reportedtumor suppressor roles, following PRP treatment.

The expression of the transcription factor FOXP1 increased 6.04 foldafter PRP treatment. In addition, the expression of TWIST2 wasup-regulated (5.2737-fold) following to PRP treatment. Another tumorsuppressor gene that was up regulated as a consequence of PRP treatmentswas THY1 (3.622 fold increase).

SPARC, an extracellular protein involved in the deposition and modellingof the extracellular matrix that is downregulated in many tumor types,was found to be up-regulated (3.611 fold) following PRP treatment. SIRT1expression was also increased 2 fold in treated cells. SIRT1 encodes amember of the sirtuin family of proteins and has been reported toinhibit proliferation of pancreatic cancer cells expressing pancreaticadenocarcinoma up-regulated factor (PAUF).

d) Expression of Genes Related to Metastasis and Invasion

The expression level of 9 genes implicated in metastasis and cellinvasion was determined following PRP treatment (FIG. 25A). The resultsshow that PRP treatment resulted in significant down-regulation of thesegenes. For example, the expression of Osteopontin (SPP1) was reduced 10fold. SPP1 is a chemokine-like, calcified ECM-associated protein thathas been reported to play a crucial role in determining the metastaticpotential of various cancers.

Signalling through platelet derived growth factor receptors (PDGFR) isinvolved in multiple tumor associated processes. The expression of PDGFRis up regulated in many tumors, for instance endothelial cells ofmetastatic tumors in models of orthotopically grown pancreatic carcinomashowed high expression of PDGFR. Following PRP treatment, expression ofPDGFRB was reduced 3.1093-fold, indicating that PRP has ananti-metastatic effect. In addition, expression of the gene FN1 thatencodes fibronectin, was also significantly reduced after PRP treatment(3.2 fold down-regulation). Fibronectin is involved in cell adhesion andmigration processes including metastasis.

E) EXPRESSION OF GENES RELATED TO CELL ADHESION

FIG. 25B shows the up-regulation of genes involved in cell adhesion. Theexpression of E-Cadherin and β-catenin was increased (4.3 and 4-fold,respectively) following PRP treatment, in agreement with previousstudies. E-Cadherin and β-catenin have known roles in EMT. The resultsindicate that PRP inhibits EMT by directly enhancing expression ofE-cadherin and β-catenin in expression in CSCs.

f) Expression of Genes Encoding Cytokines

The impact of PRP treatment on the expression of genes encodingcytokines in pancreatic CSCs was investigated. FIG. 26 shows thedown-regulation of genes encoding cytokines associated with cellproliferation, migration and invasion in cancer.

IL-8 and IL-8 receptors have been shown to be over-expressed inpancreatic cancer but suppressed in normal pancreatic tissues,suggesting that IL-8 plays an important role in the invasiveness ofhuman pancreatic cancer. Furthermore, IL-8 has also been reported topromote EMT by modulation of the tumor microenvironment. The resultspresented here show that the expression of IL-8 was down-regulated (4.5fold) following PRP treatment. The decrease in IL-8 gene expressiondetected after PRP treatment further supports the observation that PRPexerts an anti-CSC effect.

Treatment of CSCs with PRP dramatically decreased the expression ofSecreted phosphoprotein-1 (SPP1, 10 fold downregulation). SPP1 has beenreported to promote cancer cell survival and regulate tumor-associatedangiogenesis and inflammation. In addition, PRP treatment induced thedown regulation of membrane glycoprotein CD31/PECAM-1, a protein thathas been found on many tumor cells, such as human brain gliomas,carcinoma of the cervix, lung cancer, and breast cancer.

g) Expression of CSC Markers Genes

PRP treatment of pancreatic CSCs induced the down-regulation of 6 genesrecognized as CSC markers (FIG. 27 ), including CD38, CD45, Oct-04 andKLF4.

h) Expression of Genes Associated with MAPK/ERK Pathway

PRP treatment of pancreatic CSC induced the down-regulation of severalMAPK-related genes. The results are shown in FIG. 28 .

CONCLUSION

The results of the above in vitro studies show that:

-   -   1. PRP treatment destroys primary spheres and suppresses the        ability of CSCs to form secondary spheres and thereby has a        direct effect on the cancer stem cell population;    -   2. PRP has a potent anti-EMT effect in CSCs;    -   3. PRP has an anti-metastatic effect, enhancing expression of        factors which promote cell adhesion and reducing the expression        of factors associated with migration processes in CSCs;    -   4. PRP enhances expression of relevant tumor suppressor genes in        CSCs;    -   5. PRP modulates the expression of CSC marker genes;    -   6. PRP may induce differentiation of CSCs; and    -   7 PRP down-regulates the expression of cytokines that are        associated with cell proliferation, migration and invasion in        cancer.

These results reinforce the therapeutic potential of PRP as an anti-CSC,anti-EMT, anti-metastasis, and anti-tumour drug.

Example 2

Phase 1- In Vivo Study of the Antitumor Activity of PRP Against CSCs

A. Experimental Design:

-   -   1- Control group (n=10): untreated mice (inoculated with        physiological saline solution)    -   2- Prevention group: mice are treated with PRP        (Trypsinogen/Chymotrypsinogen A), prior to inoculation with CSCs        (n=10). PRP was dissolved in physiological saline solution is        administered in a single i.v. bolus injection every other day        for 3 weeks.    -   3- Prevention+Treatment group: mice are treated with PRP        (Trypsinogen/Chymotrypsinogen A), prior to and after CSC        inoculation (n=10). PRP dissolved in physiological saline        solution is administered in a single i.v. bolus injection every        other day for 3 weeks. The treatment is maintained for 3-4        months.    -   4- Treatment group: mice are treated with PRP        (Trypsinogen/Chymotrypsinogen A), only after CSC inoculation.        PRP dissolved in physiological saline solution is administered        in a single i.v. bolus injection every other day for 3-4 months.

Phase 2- Study of the Ability of PRP to Inhibit the Initiation of TumorCapacity of Pancreatic CSC

To determine whether PRP treatment can reduce the tumor-initiatingcapacity of pancreatic CSCs, the following set of experiments areperformed. Xenograft experiments are conducted in which tumor formationis induced by injection of pancreatic CSCs previously treated in vitrowith PRP. Assessment of tumor formation, and study of tumorcharacteristics is performed.

A. Experimental design:

-   -   1- Control group (n=8). NSG mice are injected with non-treated        pancreatic CSCs.    -   2- IC50 PRP group (n=8). Mice are injected with pancreatic CSCs        treated with the IC50 of PRP (Trypsinogen/Chymotrypsinogen A)        over 3-6 days.    -   3-2x IC50 PRP group (n=8). Mice are injected with pancreatic        CSCs treated with 2x the IC50 of PRP        (Trypsinogen/Chymotrypsinogen A) over 3-6 days.    -   4-5x IC50 PRP group (n=8). Mice are injected with pancreatic        CSCs treated with 5x the IC50 of PRP        (Trypsinogen/Chymotrypsinogen A) over 3-6 days.

Phase 3- Experimental Metastasis In Vivo Assay

To determine whether PRP can be used as an anti-metastasis agent, anexperimental metastasis assay is performed using immune-deficient mice.The in vivo metastasis model makes use of CSCs-L2T (enrichedsubpopulation obtained from BxPC3 transfected in vitro with luciferaseand the red fluorescent protein td-Tomato) that is directly injectedinto the circulation of NSG mice (NOD scid gamma mice) through theirtail veins. Metastatic progression is monitored by IVIS (SpectrumPreclinical In Vivo Imaging System). Mice are treated with PRP prior toand after the injection of CSC-L2T according to the followingexperimental design:

A. Experimental Design:

-   -   1- Control group (n=8): untreated mice are inoculated with        physiological saline solution.    -   2- Prevention group: Mice are treated with PRP        (Trypsinogen/Chymotrypsinogen A) prior to the inoculation of        CSCs- L2T. PRP dissolved in physiological saline solution is        administered in a single i.v. bolus injection every other day        for 3 weeks.    -   3- Prevention+Treatment group: Mice are treated with PRP        (Trypsinogen/Chymotrypsinogen A) prior to and after CSC-L2T        inoculation. PRP dissolved in physiological saline solution is        administered in a single i.v. bolus injection every other day        for 3 weeks. The treatment is maintained for 3-4 months.    -   4- Treatment group: Mice are treated with PRP        (Trypsinogen/Chymotrypsinogen A) only after inoculation with        CSCs-L2T. PRP dissolved in physiological saline solution is        administered in a single i.v. bolus injection every other day        for 3-4 months.

Material and Methods

Phase 1 and 2

Isolation of CSCs

Cancer stem-like cells from BxPC3 pancreatic cancer (adenocarcinoma)cell line are isolated using the ALDEFLUOR assay (StemCell Technologies)by fluorescence-activated cell sorting (FACS) and enriched subpopulationof CSCs will be grown with our specific sphere forming medium(PCT/ES2015/070606) in ultralow attachment plates (Corning).

In Vivo Anti-Tumor Xenograft Studies

To establish xenograft tumors six- to eight week old NSG immunodeficientmice were used. All procedures were approved by the Institutional AnimalCare and Use Committee at the University of Granada. Mice were housedand maintained at 20° C. to 24° C., 50% RH, a 14 to 10 h lightdark cyclewith food and water ad libitum.

For phase 2, pancreatic CSCs are treated for 3 and 6 days with IC₅₀, 2xIC₅₀ and 5x IC₅₀ of PRP. Cell viability is determined prior to theinoculation of PRP-treated CSCs into NSG mice model.

The BxPC3 pancreatic cancer cell line tumors were generated bysubcutaneous injections of 100-500 viable cells/mouse* using 26-gaugeneedles. Animals (n=10 per group) are then randomly assigned as controland treatment groups (groups 1-4) and are treated according to theexperimental procedure described previously (FIG. 14A and B).

Tumor weight was calculated according to the formula: TW (mg)=tumorvolume (mm³)=d2 ×D/2, where d and D are the shortest and longestdiameters, respectively. Paraffin-embedded blocks of all tumors aresectioned at 5 pm. Each sample was stained with hematoxylin and eosin(H&E) for histopathologic analysis.

In Vivo PRP Treatment

PRP (Trypsinogen/Chymotrypsinogen A) dissolved in physiological salinesolution was administered in a single i.v. bolus injection every otherday during the treatment period. The dose administered to groups 2 and 3was 83.3 mg/kg trypsinogen and 500 mg/kg chymotrypsinogen A. Trypsinogenand Chymotrypsinogen A were provided in combination and administered ina single injection. The dosing volume was 10 mL/kg. The volume of dosingsolution administered to each animal was calculated and adjusted basedon individual body weight measured immediately prior to dosing.

Treatments were administered for 3 weeks before tumour induction(prevention group) or for 3 weeks before tumour induction and for atleast 4 weeks (preferably up to 9.5 weeks) after induction(prevention+treatment group).

Preliminary Metastasis Assay

Mice were killed with a lethal dose of sodium pentobarbital (100 mg/kgbody weight). Thoracic organs are removed, the lung is washed in coldPBS and weighed. Other visceral organs are removed and inspected forpresence of metastases. Lung is fixed in formalin and embedded inparaffin. Hematoxylin and eosin (H&E)-stained lung-sections is analysedfor metastatic nodule presence.

Measurement of Effect of PRP on Tumour Growth

The effect of PRP on tumour growth was determined by calculating thetumour growth inhibition ratio, T/C. T/C was determined using twodifferent methods: the first measuring tumour volume at the end oftreatment and the second measuring the rate of tumour growth over thecourse of treatment.

In the first method, the ratio of mean volume of control tumours (C)versus the mean volume of treated tumours (T) at the final day oftreatment was calculated. T/C 0.45 is the consensus cut-off to indicateefficacy of a given treatment,

In the second method, the rate based T/C described by Hather et al.,(2014) was used. This method is based on fitting the growth curve ofeach tumour to an exponential model. The rate based T/C uses allavailable data, thus, is able to account for random differences in theinitial volume. In addition, fitting all the data reduces the effect ofmeasurement noise and allows the rate based T/C estimates to be moreprecise. A μ-value is also computed using a two sided t-test withunequal variances applied to the estimated tumour growth rates. Theμ-value calculation assumes that the estimated tumor growth rates arenormally distributed within each treatment group. A threshold of 0.4 waschosen because it is a common cut-off to determine if the antitumoractivity is sufficient to be of practical significance. The evolution oftumour growth was therefore evaluated by measuring tumour volume every 2days over 36 days and the data was analyzed using an Excel spreadsheetthat computes the rate based T/C (FIG. 18 ). The rate based T/C was 0.54(p=0.17) for pre-treated group and 0.13 (p=0.001) for the pre-treatedand treated group. Indicating that the follow up treatment with PRPsignificantly inhibits tumour growth.

Effect of PRP on Tumour Incidence and Weight

The Tumourigenesis Index (Tin) is an index that reflects the relativetumour incidence and tumour weight in a subject. The Tin can be used tomeasure malignancy or tumour aggressiveness.

Phase 3

Lentivirus Transfections

293T cells are co-transfected with a lentiviral vector encoding for thefirefly luciferase and the red fluorescent protein td-Tomato (L2T) 26, apackaging vector (psPAX2) and an envelope vector (pCMV-VSVG) usinglipofectamine transfection reagent (Life Technologies). Viral particlesproduced from 293T cells are collected and used to infect BxPC3 cells.Briefly, supernatant is recovered after 48h from 293T transfected cellsand filtered by a 0.45 pm pore membrane and added to BxPC3 plated cellssupplemented with 4 pg/mL polybrene (Sigma-Aldrich). After viralinfection, td-Tomato+ cells are selected for stable integration of thetransfects by FACS.

Experimental Lung Metastasis In Vivo Assay

BxPC3 CSCs-L2T are injected into the tail vein of 4-6 weeks old femaleNSG mice (n=8 per group). Bioluminescence is monitored by IVIS at day 0and weekly by injecting intraperitoneally 150 mg/kg of D-Luciferine(Thermo Fisher). After 4 weeks, mice are euthanized and lungs areexcised, photographed for td-Tomato expression and assayed forluciferase activity washing lungs with 150 pg/mL of D-luciferine dilutedin PBS. Excised lungs are also used for Hematoxylin and Eosin stainingand confocal microscopy analysis.

In Vivo PRP Treatment

PRP (Trypsinogen/Chymotrypsinogen A) dissolved in physiological salinesolution is administered in a single i.v. bolus injection every otherday for 3-4 months.

Histological Analysis

Lungs are immersed in 4% paraformaldehyde in 0.1 M PBS for 4 h at 4° C.,washed in 0.1M PBS and embedded in paraffin in an automatic tissueprocessor (TP1020, Leica, Germany). The paraffin blocks are cut into 4mm sections for staining. Sections are deparaffinized with xylene andhydrated with decreasing alcohol concentrations (absolute to 70%), andwill be stained with hematoxylin-eosin. Sections are then dehydratedwith increasing alcohol concentrations (95% to absolute), and clearedwith xylene and mounted with mounting medium. Observation under lightmicroscopy and digital image acquisition is performed with an invertedmicroscope (Nikon H550s).

Immunohistochemical Analysis

Lungs are immersed in 10% formaline at room temperature overnight,washed in 0.1M PBS, and preserved in 30% sucrose in PBS for 24 h. Thematerial is then soaked in OCT compound (Sakura Finitek Europe B.V.,Netherlands), frozen in liquid nitrogen and blocks stored at −40° C.until use. The OCT blocks are cut into 8 mm sections and collected onSuperFrost slides (Menzel-Glasser, Germany). The sections are hydratedwith PBS and mounted with mounting medium with DAPI. Observation underfluorescent microscopy and digital image acquisition will be performedwith a confocal microscope (Nikon Al).

Results

Preliminary results for Phase 1 experiments were obtained 4 weeks and9.5 weeks after CSC inoculation. The results demonstrate the anti-tumourefficacy of PRP (Trypsinogen/Chymotrypsinogen A) against tumours inducedby BxPC3 in human pancreatic CSCs.

FIGS. 15 to 19 show the results for the Phase 1 study, 9.5 weeks aftercommencement of the trial (i.e., 67 days after CSC inoculation).

FIG. 15 provides images of excised tumours from mice in groups 1, 2 and3.

FIG. 16 shows % tumour incidence in groups 1, 2 and 3: 9 of 10 controlmice developed a tumour after subcutaneous injection of the CSC-enrichedpopulation of pancreatic BxPC3 (this value was interpreted as 100%tumour incidence). In both groups 2 and 3 (i.e., prevention andprevention+treatment), fewer than 50% of mice developed tumours (41% ingroup 2; 50% in group 3). These results show that preventative treatmentwith PRP (Trypsinogen/Chymotrypsinogen A) prior to CSC inoculation has aclear suppressive effect on pancreatic CSC tumour engrafting.

The effect of PRP treatment in reducing tumour growth was assessed andthe results are shown in FIGS. 17 and 18 . FIG. 17 shows the volume ofeach tumour at the final day of treatment together with the T/C value(calculated using method 1) for both group 2 and 3 mice. These resultsshow that preventative treatment with PRP (group 2) andprevention+treatment (group 3) decrease tumour uptake and impair tumourgrowth compared with control non-treated animals (T/C=0.64 in group 2and T/C=0.40 in group 3).

Rate-based T/C for the two experimental groups is shown in FIG. 18 . Therate based T/C was 0.54 (p=0.17) for prevention group (group 2) and 0.13(p=0.001) for the prevention+treatment group (group 3). These resultsshow that the follow up treatment with PRP significantly inhibits tumourgrowth, evidenced by the reduced volume of tumours in this treatmentgroup. Specifically, for group 2 mice, tumour volume was only 54% of thevolume seen in tumours of control mice. In group 3 mice, the tumourvolume was only 13% that of the tumours in control mice.

Taken together, the results presented in FIGS. 17 and 18 show asignificantly reduced incidence of tumour development and reduced volumeof any tumours that do develop in mice receiving PRP as a preventativeand in mice receiving both preventative and post-CSC inoculation(treatment) PRP.

FIG. 19 shows that significant differences in Tin (p<0.01) were observedwhen mice received preventative PRP or when they received bothpreventative PRP and PRP after CSC inoculation, indicating theanti-tumourigenic effect of PRP.

FIGS. 20 and 21 shows H&E staining and Masson-trichome staining,respectively, of sections from tumours that were excised from mice inthe control, prevention and prevention+treatment groups. Therepresentative images show noticeable differences in the density of thetumours, with significantly reduced density and increased gaps betweencells observed in the tumours from both prevention andprevention+treatment groups. Further, the presence of fibrotic tissue intumours from mice receiving PRP (both prevention andprevention+treatment groups) was significantly reduced compared tocontrol mice.

In conclusion, the data demonstrate the anti-tumour effect of PRP as apreventative and treatment. PRP impaired engrafting of CSC tumours inmice and substantially reduced progression for those tumours which wereinitiated following CSC inoculation.

Example 3

Treatment of cancer using the invention may be implemented in accordancewith the following.

An individual diagnosed with colon cancer may have the bulk of thetumour mass removed by surgery (laparoscopic or conventional). Afterresection of the tumour mass the individual is administeredchymotrypsinogen and trypsinogen, optionally in combination withchemotherapy or radiotherapy. Prior to administration the presence ofcolon cancer stem cells may be determined by identifying cells in theindividual, or near the site of resection, that express markerscharacteristic of colon cancer stem cells. Exemplary markers may beCD326 and CD44 or any other markers described herein (see, Table 1). Theindividual is then monitored for recurrence at the original tumour siteand appearance of metastasis at known sites of colon cancer metastasis.The result is prevention or delay of recurrence or metastasis.

An individual diagnosed with pancreatic cancer may have the bulk of thetumour mass removed by surgery (laparoscopic or conventional). Afterresection of the tumour mass the individual is administeredchymotrypsinogen and trypsinogen, optionally in combination withchemotherapy or radiotherapy. Prior to administration, the presence ofpancreatic cancer stem cells may be determined by identifying cells inthe individual, or near the site of resection, that express markerscharacteristic of pancreatic cancer stem cells. Exemplary markers may beCD326, CD44 and CxCR4 or any other markers described herein (see, Table1). The individual is then monitored for recurrence at the originaltumour site and appearance of metastasis at known sites of pancreaticcancer metastasis. The result is prevention or delay of recurrence ormetastasis.

Similar methods may be applied to other cancers, preferably solidtumours, such as prostate cancer, breast cancer, ovarian cancer or anyother cancer described herein.

1. A method of minimising the progression of cancer in a subject who hasreceived a treatment for cancer, wherein the subject is determined tohave cancer stem cells or determined to be at risk of having cancer stemcells, the method comprising administering to the subjecttherapeutically effective amounts of chymotrypsinogen and trypsinogen,thereby minimising the progression of cancer in the subject.
 2. Themethod according to claim 1, wherein the subject is in partial orcomplete remission.
 3. The method according to claim 1, wherein themethod comprises treating minimal residual disease in a subject who hasreceived a treatment for cancer.
 4. The method according to claim 1,wherein the method comprises preventing recurrence of cancer in thesubject.
 5. A method of preventing or inhibiting metastasis of cancer ina subject who is to receive a treatment for cancer, who is receiving atreatment for cancer, or who has received a treatment for cancer,wherein the subject is determined to have cancer stem cells ordetermined to be at risk of having cancer stem cells, the methodcomprising administering to the subject therapeutically effectiveamounts of chymotrypsinogen and trypsinogen, thereby preventing orinhibiting metastasis of cancer in the subject, wherein the subject isdetermined to have cancer stem cells.
 6. The method according to claim 1wherein the treatment for cancer is selected from the group consistingof surgical excision of the tumour, radiotherapy, chemotherapy,immunotherapy or a combination thereof.
 7. (canceled)
 8. A method ofdelaying the onset of cancer in a subject, wherein the subject isdetermined to have cancer stem cells or determined to be at risk ofhaving cancer stem cells, the method comprising administeringtherapeutically effective amount of chymotrypsinogen and trypsinogen tothe subject, thereby delaying the onset of cancer in the subject.
 9. Themethod according to claim 7, wherein the subject is one who isidentified as being at risk of cancer.
 10. The method according to claim9, wherein the risk of cancer is determined on the basis of familymedical history, or biomarkers associated with risk of cancer, or acombination thereof.
 11. The method according to claim 7, wherein thesubject has not been diagnosed as having cancer.
 12. The methodaccording to claim 1 wherein the subject does not have detectable cancerat the time that the chymotrypsinogen and trypsinogen are administered.13. The method according to claim 1, wherein the chymotrypsinogen andtrypsinogen are adapted to be administered in a weight ratio ofchymotrypsinogen: trypsinogen of between greater than 1:1 but less thanor equal to 10:1.
 14. The method according to claim 13 wherein theweight ratio of chymotrypsinogen:trypsinogen is in the range of between4:1 to 8:1.
 15. The method according to claim 13 wherein the weightratio of chymotrypsinogen: trypsinogen is in the range of between 5:1 to7:1.
 16. The method according to claim 15 wherein the weight ratio ofchymotrypsinogen: trypsinogen is about 6:1.
 17. The method according toclaim 1 wherein the chymotrypsinogen and trypsinogen are administeredsimultaneously.
 18. The method according to claim 1, wherein the methodfurther comprises determining the presence of cancer stem cells in thesubject.
 19. The method according to claim 18, wherein the presence ofcancer stem cells in the subject is determined by identifying cellsexpressing one or more cancer stem cell markers.
 20. (canceled)
 21. Themethod according to claim 1 wherein the number of cancer stem cells inthe subject is reduced or prevented from increasing in number followingadministration of the chymotrypsinogen and trypsinogen. 22-38.(canceled)